The overall goal of the proposed research is to study the properties and metabolic function of hydroxylases and related enzymes in mixed-function oxidase systems in liver microsomes and in microorganisms. We have recently shown that at least four distinct forms of cytochrome P-450, as well as NADPH-cytochrome P450 reductase and phosphatidylcholine, are involved in the reconstituted liver microsomal system which hydroxylates fatty acids and steroids as well as drugs, alkanes, carcinogens, and other foreign compounds. The individual forms of cytochrome P-450 will be extensively purified and characterized by their mode of induction, immunochemical properties, chemical composition (including amino acid sequence), redox potential, and subunit composition. The catalytic activities of each form toward a variety of substrates and the role in detoxication will be determined. The interaction of substrates, reductase, and phospholipid with the cytochrome P-450 will be investigated with the goal of establishing the mechanism by which the phospholipid facilitates electron transfer and substrate hydroxylation. Attempts will be made to incorporate the purified components of the enzyme system into membranes to determine the effect of the physical environment on their activities. In candida tropicalis the cytochrome P-450, reductase, and phospholipid (lysophosphatidyl-ethanolamine) involved in the hydroxylation of alkanes, fatty acids, and drugs will be studied in a manner similar to that indicated for the liver microsomal system. In pseudomonas oleovora the rubredoxin, NADH-rubredoxin reductase, and omega-hydroxylase will be characterized, and the mechanism by which alkanes and fatty acids are hydroxylated in this enzyme system will be determined. The role of hydroperoxides will be examined. Attempts will be made to establish the nature of the active site of the hydroxylase, which is an unusual example of a mixed-function oxidase containing nonheme iron as the prosthetic group and with the iron atom liganded to atoms other than sulfur.